This project seeks to identify the ways in which changes in the cells of adult human bone marrow contribute to skeletal aging. The effects of aging on osteoblast differentiation from the human bone marrow stromal cell compartment are of importance for understanding the mechanisms and treatment of age-related bone loss and osteoporosis. Previously, we found in both two-dimensional (2D) and in three-dimensional (3D) model systems that adherent human bone marrow stromal cells (hMSCs) undergo an age-dependent decrease in capacity for differentiation to alkaline phosphatase-positive (AlkP+) cells. The first specific aim tests the hypotheses that the STRO-1-positive (+) fraction of marrow stromal cells is decreased in frequency with age and that there a decrease in differentiation to AlkP+ pre-osteoblasts and decrease in 3D bone formation in vitro. These decreases are likely to be associated with an increase in adipocyte differentiation. Methods include immuno-sorting and quantitative molecular methods to characterize the osteoblast and adipocyte lineages. The second specific aim tests the hypotheses that with aging, there is diminished signal transduction in response to osteo-anabolic agents like parathyroid hormone (PTH) and sex hormones and the third specific aim tests whether combination treatments may reverse the age-specific decrease in bone marrow stromal cell differentiation to osteoblasts. The mechanism of enhancement will be assessed, with focus on regulation of IGF-1 and on apoptosis. Age-related dysfunction in signaling and regulatory mediators will be examined. We shall determine whether there is consistency is the age-related decline in PTH signaling in hMSCs, whether this involves CREB 1 and 2 pathways, whether specific chemical inhibitors and/or CREB knockdown by siRNA technology reduce PTH signaling in hMSCs from young individuals and thus decrease IGF-I and osteoblastogenesis to levels found in elder hMSCs. This research will quantify osteoblastogenesis and bone formation by marrow cells from young and old subjects. These studies should provide valuable information on the age-dependent changes in bone marrow stromal cells, provide insights into novel strategies for rejuvenation of cellular physiology leading to improved osteogenesis, and begin to reveal mechanisms whereby these agents can promote osteoblast differentiation in cells from aged individuals.